Tripeptide derivatives with central nervous system activity and preparation thereof

ABSTRACT

The tripeptide derivatives of formula 
     
         H--L--Pro--N(R.sup.1)CH(R.sup.2)CO--Y--R.sup.3             (1) 
    
     in which R 1  is hydrogen, lower alkyl or NR 4  R 5  wherein R 4  and R 5  each are lower alkyl, R 2  is hydrogen or lower alkyl, R 3  is amino, lower alkylamino, di(lower)alkylamino or amino(lower)alkylamino and Y is one of the amino acid residues Gly or D-Ala with the proviso that when R&#39; is NR 4  R 5  wherein R 4  and R 5  are as defined herein and R 2  and Y are as defined herein, R 3  is lower alkylamino, di(lower)alkylamino or amino(lower)alkylamino, .Iadd.and with the further proviso that when R 1  is hydrogen, R 2  is hydrogen or lower alkyl and Y is Gly then R 3  is amino(lower)alkylamino, .Iaddend.and a method for their preparation are disclosed. The tripeptide derivatives of formula 1 possess central nervous system activity and methods for their use are given.

BACKGROUND OF THE INVENTION

a. Field of Invention

The present invention relates to tripeptide derivatives with centralnervous system activity, to a process for their preparation, and tointermediates therefor.

b. Description of the Prior Art

The main obstacle to the practical use of many biologically activepeptides is their brief period of action which is partly due to theirinactivation by proteolytic enzymes. An example of such a peptide is thetripeptide which is the factor inhibiting release of the melanocytestimulating hormone (MIF or MRIH).

This tripeptide was isolated from bovine hypothalamic tissue by R.M.G.Nair et al., Biochem. Biophys. Res. Commun., 43, 1376 (1971) and itsstructure was established as the C-terminal tripeptide of oxytocin:H-L-prolyl-L-leucyl-glycinamide.

This tripeptide was shown to exert an action on the central nervoussystem (CNS). The tripeptide potentiates the behavioral effects of(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) as shown by N. P. Plotnikoff etal., Life Sciences, 10, part 1, 1279 (1971) and E. Friedman et al.,Science, 182, 831 (1973). The tripeptide antagonizes the effects ofoxotremorine [N. P. Plotnikoff et al., Proc. Soc. Exp. Biol. Med., 140,811 (1972)] and reverses the sedative effects of deserpidine in mice andmonkeys [N. P. Plotnikoff et al., Neuroendocrinology, 11, 67 (1973)]. Onthe basis of the above biological activities A. V. Schally et al.,Science 179,341 (1973) have suggested that the tripeptideH--Pro--Leu--Gly--NH₂ could be useful in the treatment of patientssuffering from depression or parkinsonism.

Since the elucidation of the structure of the above tripeptide, alimited number of analogs of this peptide have been synthesized by M. E.Celis et al., Febs Letters, 27, 327 (1972) and S. Castensson et al.,Febs Letters, 44, 101 (1974). However, the natural tripeptide and theanalogs known to date have the disadvantage of possessing a shortduration of action due to rapid inactivation in the mammalian body andT. W. Redding et al., Neuroendocrinology, 11, 92 (1973) havedemonstrated that the first step in the inactivation of the naturaltripeptide appears to be proteolytic cleavage of the Pro-Leu bond withformation of proline and leucyl-glycinamide.

Accordingly, analogs of the natural tripeptide having a greaterresistance to protease hydrolysis while retaining the CNS activity ofthe natural tripeptide are of interest. The present invention disclosesnovel analogs of the natural tripeptide in which the leucyl and glycylamino acid residues may be replaced and the peptide linkage and theterminal amide may be substituted.

In addition, an unique and straightforward process for preparing thesetripeptide derivatives is disclosed.

SUMMARY OF THE INVENTION

The peptide derivatives of this invention are represented by the generalformula 1. ##STR1## in which R¹ is hydrogen, lower alkyl or NR⁴ R⁵wherein R⁴ and R⁵ each are lower alkyl; R² is hydrogen or lower alkyl;R³ is amino, lower alkylamino, di(lower)alkylamino oramino(lower)alkylamino and Y is one of the amino acid residues Gly orD--Ala with the proviso that when R¹ is NR⁴ R⁵ wherein R⁴ and R⁵ are asdefined herein and R² and Y are as defined herein, R³ is loweralkylamino, di(lower)alkylamino or amino(lower)alkylamino.Iadd., andwith the further proviso that when R¹ is hydrogen, R² is hydrogen orlower alkyl and Y is Gly then R³ is amino(lower)alkylamino. .Iaddend.

One embodiment of the process of this invention proceeds through aseries of intermediates of the formula (2)

    R.sup.6 --L--Pro--N(R.sup.1)CH(R.sup.2)CO--Gly--R.sup.7    ( 2)

in which R¹ is lower alkyl or NR⁴ R⁵ in which R⁴ and R⁵ are each loweralkyl, R² is hydrogen or lower alkyl, R⁶ is an amino protective group asused in peptide synthesis, and R⁷ is hydroxyl, lower alkoxy, amino,lower alkylamino, di(lower)alkylamino, amino(lower)alkylamino, orprotected amino(lower)alkylamino.

The process comprises the condensation of an enamine or hydrazone offormula 3, R¹ N=CHR², in which R¹ and R² are as defined immediatelyabove with an amino acid of the formula R⁶ --L--Pro--OH in which R⁶ isas defined immediately above in the presence of an .[.isonitriie.]..Iadd.isonitrile .Iaddend.of the formula CNCH₂ COR⁷ in which R⁷ is loweralkoxy, to obtain the corresponding intermediate of formula 2 in whichR¹, R², and R⁶ are as defined immediately above and R⁷ is lower alkoxy.Transformation of said last-named compound by standard procedures knownto be effective for transforming lower alkyl ester into thecorresponding amide or substituted amide gives the correspondingcompound of formula 2 in which R⁷ is amino, lower alkylamino,di(lower)alkylamino, or protected amino(lower)alkylamino, and.[.removed.]. .Iadd.removal .Iaddend.of the protective groups(s) givesthe corresponding compound of formula 1.

The preferred process used in the above embodiment comprises thecondensation of a compound of formula 3 with an amino protected prolineof formula R⁶ --L--Pro--OH in which R⁶ is as defined herein, in thepresence of an isonitrile of formula CNCH₂ COR⁷ in which R⁷ is asdefined herein to obtain the corresponding intermediate of formula 2 inwhich R¹, R², R⁶ and R⁷ are as defined immediately above, followed bytreatment of said intermediate 2 with ammonia to obtain thecorresponding amide; and removing the protective group R⁶ to obtain thecorresponding peptide derivative of formula 1 in which R¹ and R² are asdefined immediately above, R³ is amino and Y is the amino acid residueGly.

Alternatively, the intermediate of formula 2 in which R¹, R², R⁶ and R⁷are as defined immediately above is treated with a hydrolyzing agent toobtain the corresponding acid of formula 2 in which R¹, R² and R⁶ are asdefined immediately above and R⁷ is hydroxyl. The latter acid is treatedwith an agent generally useful in peptide chemistry for activating acarboxyl group, and condensing the activated compound with a loweralkylamine, di(lower)alkylamine or mono protected amino(lower)alkylaminegives the corresponding intermediate of formula 2 in which R¹, R² and R⁶are as defined immediately above and R⁷ is lower alkylamino,di(lower)alkylamino or protected amino(lower)alkylamino. The protectivegroup(s) in said last-named compound are removed to obtain thecorresponding tripeptide derivative of formula 1 in which R¹ and R² areas defined immediately above, R³ is alkylamino, di(lower)alkylamino, oramino(lower)alkylamino, and Y is the amino acid residue Gly.

A further alternate embodiment of the process of this inventioncomprises the preparation of tripeptide derivatives of formula 1 by thestepwise addition of amino acids. Preferred compounds of formula 1obtained by this alternate embodiment are those in which R¹ is hydrogenor lower alkyl, R² is hydrogen or lower alkyl, preferably an amino acidside chain, R³ is amino, lower alkylamino, di(lower)alkylamino oramino(lower)alkylamino and Y is the amino acid residue Gly or D--Ala.Iadd.with the proviso that when R¹ is hydrogen, R² is hydrogen or loweralkyl and Y is Gly then R³ is amino(lower)alkylamino. .Iaddend.

The tripeptide derivative of formula 1 in which R¹ is CH₃, R² is CH₂CH(CH₃)₂, i.e. the amino acid side chain of L-leucine, R³ is NH₂ and Yis the amino acid residue D--Ala is readily prepared by coupling anactivated ester of benzyloxycarbonyl-L-(N-methyl)leucine with .[.D-alanice.]. .Iadd.D-alanine .Iaddend.methyl ester to obtain the dipeptideof formula Z--L--(N--Me)Leu--D--Ala--OMe. The amino protecting group (Z)of the latter compound is removed, followed by coupling with anactivated ester of benzyloxycarbonyl-L-proline to give the tripeptide offormula Z--L--Pro--L--(N--Me)Leu--D--Ala--OMe. The latter compound, whensubjected to the action of ammonia in an inert organic solvent, givesthe tripeptide of formula Z--L--Pro--L--(N--Me)Leu--D--Ala--NH₂. Theamino protecting group (Z) of the latter compound is removed to obtainthe corresponding tripeptide derivative of formula 1 in which R¹ is CH₃,viz., H--L--Pro--L--(N--Me)Leu--D--Ala--NH₂ .

The tripeptide of formula 1 in which R¹ is hydrogen, R² is CH₂ CH(CH₃)₂,R³ is NH(CH₂)₄ NH₂ and Y is the amino acid residue Gly is readilyprepared by subjecting the tripeptide of formulaZ--L--Pro--L--Leu--Gly--OEt to hydrolysis to obtain the correspondingacid of formula Z--L--Pro--L--Leu--Gly--OH. The carboxyl of saidlast-named compound is activated and condensed with a mono protectedamino-1,4-diaminobutane, for example H₂ N(CH₂)₄ NHBoc to give thecorresponding tripeptide of formula Z--L--Pro--L--Leu--Gly--NH(CH₂)₄NH--Boc. The amino protecting groups of the latter compound are removedto obtain the corresponding tripeptide derivative of formula 1, viz.,L--Pro--L--Leu--Gly--NH(CH₂)₄ NH₂.

DETAILS OF THE INVENTION

The term "lower alkyl" as used herein contemplates straight chain alkylradicals containing from one to six carbon atoms and branched chainalkyl radicals containing three to four carbon atoms excluding t-butyland includes methyl(Me), ethyl(Et), propyl, isopropyl, butyl, isobutyl,pentyl and the like.

In general the abbreviations used herein for designating the amino acidsand the protective groups are based on recommendations of the IUPAC-IUBCommission on Biochemical Nomenclature see Biochemistry, 11, 1726-1732(1972). For instance Pro, Leu, Ala and Gly represent "residues" ofproline, leucine, alanine and glycine, respectively. By the residue ismeant a radical derived from the corresponding α-amino acid byeliminating the OH portion of the carboxyl group and the H portion ofthe α-amino group. The term "amino acid side chain" is that part of anamino acid exclusive of the --CH(NH₂)COOH portion, as defined by K. D.Kopple, "Peptides and Amino Acids", W. A. Benjamin Inc., New York andAmsterdam, 1966, pages 2 and 33; examples of such side chains of thecommon amino acids are --CH₂ CH(CH₃)₂ (the side chain of leucine) or H--(the side chain of glycine).

The configuration of the amino acids and amino acid residues herein aredesignated by the appropriate symbols D, L or DL, furthermore when theconfiguration is not designated the amino acid or residue can have theconfiguration D, L or DL. It will be noted that the structure of some ofthe compounds of this invention includes asymmetric carbon atoms. It isto be understood accordingly that the isomers arising from suchasymmetry are included within the scope of this invention. Such isomersare obtained in substantially pure form by classical separationtechniques and by sterically controlled synthesis and have arbitrarilybeen named as isomers A or B, respectively.

A number of procedures or techniques for the preparation of peptideshave hitherto been well established and found in general textbooks ofpeptide chemistry; for example K. D. Kopple, supra, pp. 33-51 and E.Schroder and K. L. Lubke, "The Peptides"; Vol. 1; Academic Press, NewYork, 1965, pp. 3-128. For instance, the functional groups which are notinvolved in the peptide bond formation reaction are optionally protectedby a protecting group or groups prior to the condensation reaction.Examples of protecting groups for an amino function of a peptide oramino acid not involved in the peptide bond formation are: thealkoxycarbonyls which include benzyloxycarbonyl (represented by Z),t-butoxycarbonyl (Boc), or α,α-dimethyl-3,4-dimethoxybenzyloxycarbonyl(Ddz); the acyl type protecting groups which include triphenylmethyl orbenzyl. The preferred protecting groups are benzyloxycarbonyl andt-butoxycarbonyl. The carboxylic acid function of a peptide or aminoacid can be considered protected by a lower alkyl or lower aralkyl esterwhich includes methyl (represented by OMe), ethyl (OEt), benzyl (OBzl)or tert-butyl (OBu^(t)).

To promote facile condensation of a peptide carboxyl group with a freeamino group of another peptide to form a new peptide bond, the terminalcarboxyl group must be activated. Descriptions of suchcarboxyl-activating groups are included in the general textbooks ofpeptide chemistry by Kopple, or Schroder and Lubke, cited above.Examples of the activated form of a terminal carboxyl are acid chloride,anhydride, azide, imidazolide, activated ester or O-acyl urea of adialkylcarboxdiimide. The following activated esters have proved to beparticularly suitable in the process of this invention:2,4,5-trichlorophenyl (represented by OTcp), pentachlorophenyl (OPcp),p-nitrophenyl-(ONp), or.[.L-benzotriazolyl.]..Iadd.1-benzotriazolyl.Iaddend.; the succinimidoderivative is also useful for this purpose.

The terms "peptide, dipeptide, tripeptide, and the like" used herein arenot limited to refer to the respective parent peptides but also are usedin reference to modified peptides having functionalized or protectinggroups. The term "peptide" as used herein is used in reference to apeptide with one to three amino acid residues.

The term "mineral acid" as used herein contemplates the strong inorganicacids and includes hydrochloric, hydrobromic, sulfuric, and phosphoricacid. When the term is used in conjunction with an anhydrous system,anhydrous hydrochloric acid is the preferred mineral acid.

The term "mildly acidic conditions" as used herein contemplatesconditions in which a dilute aqueous solution of an organic acid, forexample 30-90%, preferably 70-80%, aqueous formic, acetic or propionicacid, or 1 to 10% aqueous trifluoroacetic acid is a principal componentof the reaction medium, usually at 20°-50° C.

The term "moderately acidic conditions" as used herein contemplatesconditions in which concentrated organic acids or aqueous solutions ofthe mineral acids are used as a principal component of the reactionmedium at termperatures ranging from about -30° to 30° C. Examples ofpreferred conditions in this case include the use of 50 to 100%trifluoroacetic acid at 0° to 30° C., 0.1 to 12N hydrochloric acid at-30° to 10° C. or 0.1 to 6N hydrogen chloride in an anhydrous inertorganic solvent.

The term "organic base" as used herein includes triethylamine,N-ethylmorpholine and N-ethyldiisopropylamine.

The term "strong base" as used herein contemplates both organic bases,as described above, and strong inorganic bases including the hydroxidesand carbonates of sodium and potassium.

The tripeptides of this .[.inventin.]. .Iadd.invention .Iaddend.areobtained in the form of the free base or as an acid addition saltdirectly from the process of this invention. The tripeptides in the formof the free base are readily obtained from the corresponding acidaddition salt by conventional methods, for example the free base isreadily obtained from the acetic acid addition salt by repeatedlyophilization of the latter salt from aqueous solution. The acetic acidaddition salt is readily obtained from another acid addition salt bytreatment with the appropriate ion exchange resin in the mannerhereinafter disclosed. The tripeptides of this invention are obtained inthe form of a pharmaceutically acceptable acid addition salt eitherdirectly from the process of this invention or by reacting thetripeptide with one or more equivalents of the appropriate acid.Examples of preferred non toxic salts are those with pharmaceuticallyacceptable organic acids, e.g. acetic, lactic, succinic, benzoic,salicyclic, methanesulfonic, toluenesulfonic, or pamoic acid, as well aspolymeric acids such as tannic acid or carboxymethyl cellulose, andsalts with inorganic acids such as the hydrohalic acids, e.g.hydrochloric acid, or sulfuric acid, or phosphoric acid. It should benoted that the tripeptides of this invention have one or two basicnitrogens giving rise to addition salts with one to possibly twoequivalents of acid. If desired a particular acid addition salt isconverted into another acid addition salt, e.g., a salt with a nontoxic, pharmaceutically acceptable acid, by treatment with theappropriate ion exchange resin in the manner described by R. A.Boissonas, et al., Helv. Chim. Acta, 43, 1349 (1960). Suitable ionexchange resins are cellulose based cation exchangers, for examplecarboxymethylcellulose, or chemically modified, cross-linked dextrancation exchangers, for example, those of the Sephadex C type, andstrongly basic anion exchange resins, for example those listed in J. P.Greenstein and M. Winitz "Chemistry of the Amino Acids", John Wiley andSons, Inc., New York and London, 1961, Vol. 3, p. 1456.

The tripeptide derivatives produced by the process of this invention, aswell as their corresponding pharmaceutically acceptable salts, areuseful because they show the pharmacological activities upon the CNS ofwarm-blooded animals possessed by the natural tripeptideH-L-prolyl-L-leucyl-glycinamide, and at least one of the compounds ofthis invention shows activities greater than those of the naturaltripeptide. For example, the compounds of this invention potentiate theeffects of L-DOPA when tested by the method G. M. Everett, Proc. Firstinternat. Sympos. Antidepr. Drugs, Excerpta Medica Internat. Congr.Series no. 122, 164 (1966) in the modification described by N. P.Olotnikoff et al., Life Sciences Vol. 10, Part 1, p. 1279 (1971). Thetripeptide derivatives of formula 1 also antagonize fluphenazine-inducedcatalepsy in rats, an animal model particularly suitable for screeningcompounds useful in the management of Parkinson-like .[.diorders.]..Iadd.disorders.Iaddend., and they cause reversal of the sedative effectof deserpidine. The tripeptide derivatives of this invention have aprolonged duration of action and are useful for treating or managingcentral nervous system disorders, especially Parkinsonism or mentaldepression, in warm-blooded animals. When a tripeptide of this inventionor a slat is employed for such treatment or management, it isadministered systemically, preferably parenterally, in combination witha pharmaceutically acceptable liquid or solid carrier. The peptides offormula 1 have a lower order of toxicity. The proportion of thetripeptide or salt thereof is determined by its solubility in the givencarrier, by the given carrier, by the chosen route of administration andby standard biological practice. For parenteral administration toanimals the tripeptide or a salt thereof is used in a sterile aqueoussolution which may also contain other solutes such as buffers orpreservatives, as well as sufficient pharmaceutically acceptable saltsor glucose to make the solution isotonic. The dosage will vary with theform of administration and with the particular species of animal to betreated and is preferably kept at a level of from 0.05 mg to 20 mg perkilogram body weight. However, a dosage level in the range of from about0.05 mg to about 2 mg per kilogram body weight is most desirablyemployed in order to achieve effective results.

For oral administration to animals the dosage of the tripeptide or asalt thereof is preferably kept at a level of from 0.25 mg to 100 mg perkilogram body weight, and the compound is formulated in unit dosage formwith pharmaceutically acceptable carriers. The tripeptide or a saltthereof may also be administered directly to the interior surface of themouth, for example in one of the dosage forms described in U.S. Pat.application Ser. No. 567,788, filed Apr. 14, 1975.

The tripeptide or a salt thereof may also be administered in one of thelong acting, slow-release or depot dosage forms described below,preferably by intramuscular injection or by implantation. Such dosageforms are designed to release from about 0.05 mg to about 2 mg perkilogram body weight per day.

It is often desirable to administer a tripeptide of formula 1continuously over prolonged periods of time in long-acting,slow-release, or depot dosage forms. Such dosage forms may eithercontain a pharmaceutically acceptable salt of the tripeptide having alow degree of solubility in body fluids, for example one of those saltsdescribed below, or they may contain the tripeptide in the form of awater-soluble salt together with a protective carrier which preventsrapid release. In the latter case, for example, the tripeptide may beformulated with a nonantigenic partially hydrolyzed gelatin in the formof a viscous liquid; or the tripeptide may be absorbed on apharmaceutically acceptable solid carrier, for example, zinc hydroxide,and may be administered in suspension in a pharmaceutically acceptableliquid vehicle; or the tripeptide may be formulated in gels orsuspensions with a protective non-antigenic hydrocolloid, for examplesodium carboxymethylcellulose, polyvinylpyrrolidone, sodium alginate,gelatine, polygalacturonic acids, for example, pectin, or certainmucopolysaccharides, together with aqueous or non-aqueouspharmaceutically acceptable liquid vehicles, preservatives, orsurfactants. Examples of such formulations are found in standardpharmaceutical texts, e.g. in Remington's Pharmaceutical Sciences, 14thEd., Mack Publishing Co., Easton, Pa., 1970. Long-acting, slow-releasepreparations of the tripeptide produced according to the process of this.[.inention.]. .Iadd.invention .Iaddend.may also be obtained bymicroencapsulation in a pharmaceutically acceptable coating, for examplegelatine, polyvinyl alcohol or ethyl cellulose. Further examples ofcoating materials and of the processes used for microencapsulation aredescribed by J. A. Herbig in "Encyclopedia of Chemical Technology", Vol.13, 2nd Ed., Wiley, New York 1967, pp. 436-456. Such formulations, aswell as suspensions of salts of the tripeptide which are only sparinglysoluble in body fluids, for example the salt with pamoic acid, aredesigned to release from about 0.05 mg to about 2 mg of the activecompound per kilogram body weight per day, and are preferablyadministered by intramuscular injection. Alternatively, some of thesolid dosage forms listed above, for example certain sparinglywater-soluble salts or dispersions in or adsorbates on solid carriers ofslats of the agent, for example dispersions in a neutral hydrogel of apolymer of ethylene glycol methacrylate or similar monomers cross-linkedas described in U.S. Pat. No. 3,551,556 may also be formulated in theform of pellets releasing about the same amounts as shown above and maybe implanted subcutaneously or intramuscularly.

Process

The process of this invention is illustrated by the followingdescription of preferred embodiments.

In the practice of an embodiment of the process of this invention thefirst group of requisite starting materials, the enamines or hydrazonesof formula 3, R¹ N=CHR² in which .[.Ris.]. .Iadd.R is .Iaddend.loweralkyl or NR⁴ R⁵ in which R⁴ and R⁵ are lower alkyl and R² is hydrogen orlower alkyl are prepared by condensing an appropriately substitutedamine of formula R¹ NH₂ or a hydrazine of formula R⁴ R⁵ NNH₂ in whichR¹, R⁴ R⁵ are as defined immediately above, with an aldehyde of formulaR² CHO in which R² is as defined above.

The amines of formula R₁ NH₂ or the hydrazines of formula R⁴ R⁵ NNH₂ areeither known or they are prepared by known methods. Likewise, thealdehydes of formula R² CHO are known and most are commerciallyavailable.

The condensation of the amine of formula R¹ NH₂ or of the hydrazine offormula R⁴ R⁵ NNH₂ with the aldehyde of formula R² CHO is preferablycarried out in an inert solvent at an elevated temperature, at or nearthe reflux temperature of the mixture. Either an anhydrous,water-immiscible hydrocarbon solvent, for example, benzene or toluene,with concomitant physical removal of water as it is being formed, forexample, by means of a Dean-Stark water separator, or a lower alkanolsolvent, for example, ethanol, propanol, or isopropanol may be employed.Thereafter, evaporation of the solvent and purification of the residue,for example by distillation or crystallization, yields the correspondingenamine or hydrazone of formula 3. Alternatively, the desired enamine orhydrazone may be prepared in situ during the course of the key reaction,see below.

The second group of of requisite starting materials, the amino protectedacids of formula R⁶ --L--Pro--OH in which R⁶ is as defined hereinbeforeare known. For example, t-butoxycarbonyl-L-pyoline (Boc--L--Pro--OH) andbenzyloxycarbonyl-L-proline (Z--L--Pro--OH) are described by G. R.Anderson and A. C. McGregor, J. Amer. Chem. Soc., 79, 6180 (1957) and W.Grassmann and E. Wusch, Chem. Ber., 91, 462 (1958), respectively.

The third group of requisite starting materials, the isonitriles offormula CNCH₂ COR⁷ in which R⁷ is lower alkoxy with 1-3 carbon atoms areeither known, e.g. ethyl isocyanoacetate is descried by R. Appel et al.,Angew. Chem., Int. ed., 10, 132 (1972) or are easily prepared by knownmethods.

Next, in a key reaction of the process of this invention theaforementioned enamine or hydrazone of formula 3, or alternatively thedesired anamine or hydrazone prepared in situ from the respective amineor hydrazine and aldehyde, is condensed with the acid of formula R⁶--L--Pro--OH and the isonitrile of formula CNCH₂ COR⁷ to yield thecorresponding intermediate of the formula R⁶--L--Pro--N(R¹)--CH(R²)CO--Gly--R⁷ (2) in which R¹, R², R⁶ and R⁷ are asdefined immediately above.

Although not critical, it is preferable to use approximately equimolaramounts of the requisite starting materials for this condensation. Thecondensation is effected most conveniently in an inert solvent, forexample, in halogenated hydrocarbons including methylene chloride,chloroform, and carbon tetrachloride; in ethers and cyclic ethersincluding dioxane, diethyl ether and tetrahydrofuran; or in loweraliphatic alcohols including methanol, ethanol and propanol. However,when the starting materials are mutually soluble or the mixture thereofbecomes liquid during the course of the condensation the solvent may beomitted without any deleterious effects.

The temperature and duration of the condensation are also not critical.The reaction may be performed at temperatures ranging from -20° to 100°C.; however, a range from 10° to 40° C. is most convenient. The reactiontime may be varied and depends on the reactivity of the various startingmaterials; however, reaction times from 15 minutes to several days areemployed generally, with six hours to two days being preferred.

Thereafter, the intermediate of formula 2 in which R¹, R², R⁶ and R⁷ areas defined immediately above is isolated and purified according tostandard procedures. For instance the product is extracted with awater-immiscible solvent and, if needed, purified by chromatography andcrystallization.

It will be apparent to those skilled in the art that the amino acidresidue represented in formula 2 by --N(R¹)CH(R²)CO--to as obtained inthis reaction must be racemic, and it is therefore designated in thisApplication by the prefix DL, except when R² is hydrogen and the aboveamino acid residue represents the amino acid residue of glycine. Also,it is apparent that the intermediate of formula 2 exists in the form oftwo geometric isomers which may be separated, for example bychromatography on silica gel. For convenience, these two isomers aredesignated arbitrarily as isomers A and B. Thereafter either theseparate isomers or the mixtures thereof are transformed to thecorresponding peptide derivatives of formula 1 in the manner disclosedbelow.

Said intermediate of formula 2 is subjected to amidation, to obtain theintermediate amide of formula 2 in which R¹, R², R⁶ are as definedimmediately above and R⁷ is NH₂. Preferred conditions for this amidationinclude treating said intermediate with a substantially saturatedsolution of ammonia in an inert solvent, for example, methanol, ethanolor tetrahydrofuran, at 0° to 20° C. for 6 hours to 4 days. If desiredthe corresponding amide thus obtained may be separated into two isomersat this stage. This separation is effected conveniently bychromatography on silica gel.

The above amide is then treated with a deprotecting agent to obtain thecorresponding tripeptide derivative of formula 1 in which R¹ is loweralkyl or NR⁴ R⁵ wherein R⁴ and R⁵ each are lower alkyl, R² is hydrogenor lower alkyl, R³ is amino and Y is the amino acid residue Gly. Thetripeptide derivative of formula 1 in which R¹ is NR⁴ R⁵ wherein R⁴ andR⁵ each are lower alkyl, R² is hydrogen or lower alkyl, R³ is amino andY is the amino acid residue Gly has been disclosed in the copending U.S.Pat. application Ser. No. 330,352, filed Feb. 7, 1973.

The above deprotecting reaction when R⁶ is benzyloxycarbonyl (Z) isachieved conveniently by subjecting said amide to hydrogenation in thepresence of a noble metal catalyst. Preferred noble metal catalysts foreffecting the above and other hydrogenations in the process of thisinvention include those of palladium and platinum, for example, 5%palladium on charcoal or 5% platinum on charcoal; the hydrogenationiself being performed in an inert solvent, for example, acetic acid,methanol, ethyl acetate and the like. In the present instance thehydrogenation is preferably carried out with 5% palladium on charcoal inmethanol whereby the hydrogenation product, the corresponding tripeptidederivative of formula 1, is obtained in the form of the free base byseparating the catalyst from the reaction mixture and evaporating thesolvent. The deprotecting reaction when R⁶ is t-butoxycarbonyl, isachieved conveniently by subjecting said amide to moderately acidicconditions to obtain the corresponding deprotected compound. Inpractising the above deprotecting reaction it is convenient to dissolvesaid amide in an excess of trifluoracetic acid or in an inert organicsolvent, for example, ethyl acetate or tetrahydrofuran substantiallysaturated with anhydrous hydrogen chloride. After completion of thereaction, evaporation gives directly the aforementioned deprotectedtripeptide derivative of formula 1 in the form of the acid addition saltof the corresponding acid. The latter acid addition salt may beconverted to its corresponding tripeptide derivative of formula 1 in theform of the free base by standard means.

The tripeptide derivative of formula 1, is NR⁴ R⁵ wherein R⁴ and R⁵ achare lower alkyl; R² is CH₂ CH(CH₃)₂ ; R³ is NH(CH₂)₄ NH₂ and Y is theamino acid residue Gly, is prepared by treating the intermediate offormula 2 in which R¹ is NR⁴ R⁵ wherein R⁴ and R⁵ each are lower alkyland R² is CH₂ CH(CH₃)₂, R⁶ is an amino protecting group and R⁷ is loweralkoxy with a hydrolyzing agent to obtain the corresponding acid of theintermediate of formula 2 in which R¹, R² and R⁶ are as defined hereinand R⁷ is hydroxyl. For basic hydrolysis a preferred method involvessubjecting the lower alkyl ester to the action of a strong base, forexample, sodium or potassium hydroxide, in the presence of sufficientwater to effect hydrolysis of the ester. The hydrolysis is performedusing a suitable solvent, for example, methanol or ethanol. The reactionmixture is maintained at a temperature of from 0° to 50° C., preferably20° to 30° C., until hydrolysis occurs. Usually from 10 to 30 hours issufficient for this hydrolysis. The reaction mixture is then renderedacidic with an acid, for example, hydrochloric acid, sulfuric acid andthe like, and extracted with a substantially water immiscible organicsolvent, preferably chloroform. The organic solvent is evaporated toobtain said corresponding acid.

The above corresponding acid is treated with a reagent for transformingan amino or peptide acid to a corresponding compound having an activatedcarboxyl and condensed with mono(t-butoxycarbonyl)-1,4-diaminobutane. Apreferred method in practising this reaction is effected by reacting thecorresponding acid with an approximately equimolar amount of N,N¹-carbonyldiimidazole in an inert organic solvent, preferablydimethylformamide, at about -20° to -10° C. for about 20 to 50 minutes.The compound having an activated carboxyl is treated with a solution ofa substantially equimolar amount of monomono(t-butoxycarbonyl)-1,4-diaminobutane hydrochloride, described by R.Geiger, Annalen, 750, 165 (1971), and an organic base, preferablytriethylamine, in an inert organic solvent, preferablydimethylformamide. The mixture is stirred at about 20° to 30° C. forabout 15 to 30 hours and evaporated. The residue is taken up in asubstantially water immiscible solvent, preferably ethyl acetate, washedand evaporated. The residue is subjected to chromatography on silica gelusing mixtures of halogenated hydrocarbon, lower alkanols, and organicbases, preferably chloroform-methanol-pyridine for elution to obtain thetwo Isomers A and B of the amino protected intermediate of formula 2.[.Z--L--Pro--N(NR^(4R5))CH[CH₂ CH--(CH₃)₂ ]CO--Gly--NH(CH₂)₄--NH--Boc.]. .Iadd.Z--L--Pro--N(NR⁴ R⁵)CH[CH₂ CH--(CH₃)₂]CO--Gly--NH(CH₂)₄ --NH--Boc .Iaddend.in which R⁴ and R⁵ are as definedherein.

Thereafter the protective groups Z and Boc are removed from saidlast-named compound to obtain the tripeptide derivative ofH--L--Pro--N(NR⁴ R₅)CH[CH₂ CH(CH₃)₂ ]CO-- Gly--NH(CH₂)₄ NH₂, i.e. thecompound of formula 1 in which R¹ is NR⁴ R⁵ wherein R⁴ and R⁵ each arelower alkyl, R² is CH₂ CH(CH₃)₂, R³ is NH(CH₂)₄ NH₂ and Y is the aminoacid residue Gly.

The two amino protective groups Z and Boc may be removed simultaneously,for example, by using a strong acid, i.e. hydrobromic acid in aceticacid or hydrofluoric acid, or preferably the deprotection is achieved ina stepwise manner. The amino protected intermediate described above issubjected to hydrogenation as shown above in the presence of a noblemetal catalyst in an inert solvent, preferably 5% palladium on charcoalin acetic acid, in order to remove the amino protecting group,benzyloxycarbonyl(Z). The mixture is filtered and cooled to about 0° to10° C. and treated with a moderately strong acid to remove the remainingamino protecting group, t-butoxycarbonyl(Boc). An example of such anacid is hydrogen chloride. The anhydrous acid is either added directlyto the above filtrate or a solution of the acid in an inert organicsolvent, for example, ethyl acetate, tetrahydrofuran and the like, isadded. The mixture is stirred at about 0° to 25° C. for about one tothree hours and evaporated. The residue is subjected to chromatographyon a column of a cross-linked dextran absorbent (Sephadex LH-20) usingmethanol as eluant to obtain said last-named tripeptide derivative offormula 1 as the hydrochloric acid addition salt. The free base of thesaid last-named tripeptide derivative of formula 1 is obtained byconventional methods, for example by conversion to the acetate saltfollowed by lyophilization.

An alternative embodiment of the process of this invention is thepreparation of tripeptide derivatives of formula 1 by the stepwiseaddition of amino acids.

The intermediate described above, Boc--L--Pro--L--(N--Me)Leu--Gly--OEt,i.e. the compound of formula 2 in which R¹ is CH₃, R² is CH₂ CH(CH₃)₂and R⁷ is OEt is also prepared readily by the stepwise addition of aminoacids.

In a preferred embodiment of this alternative preparation of saidlast-named intermediate of formula 2, the starting materialbenzyloxycarbonyl--L--(N-methyl)leucine, described by J. R. Coggins andN. L. Benoiton, Can. J. Chem., 49, 1968 (1971), is converted to itsactivated 2,4,5-trichlorophenyl ester by treating said starting materialwith substantially one molar equivalent of 2,4,5-trichlorophenol in aninert organic solvent, preferably methylene chloride or tetrahydrofuran,in the presence of 1.1 to 1.5 molar equivalents ofdicyclohexylcarbodiimide at -20° to 0° to C. for about 45 to 75 minutesand then at 20° to 30° C. for one to three hours. The activated ester,i.e. the 2,4,5-trichlorophenyl ester of Z--L--(N--Me)Leu--OH, is thencoupled with a substantially equimolar amount of glycine ethyl esterhydrochloride in the presence of an organic base, preferablyN-ethylmorpholine, in an inert organic solvent, preferablydimethylformamide at 0° to 30° C. for 10 to 24 hours to obtain thedipeptide of formula Z--L--(N--Me)Leu--Gly--OEt. Thereafter the aminoprotecting group, Z of said last-named compound is removed, preferablyby dissolving the compound in acetic acid containing about three molarequivalents of hydrobromic acid and stirring at 20° to 30° C. for fourto five hours to obtain the dipeptide of formulaH--L(N--Me)Leu--Gly--OEt as the hydrobromic acid addition salt. Saidlast-named dipeptide is coupled with the 1-benzotriazolyl ester ofBoc--L--Pro--OH, which is prepared by combining Boc--Pro--OH with one totwo molar equivalents of 1-hydroxybenzotriazole and 1.1. to 1.5 molarequivalents of dicylohexylcarbodiimide in an inert organic solvent,preferably tetrahydrofuran, at about -5° to 0° C. The mixture is stirredat about -5° to 0° C. for about 1 hour and then at 20° to 30° C. for anadditional hour. This solution containing the 1-benzotriazolyl ester ofBoc--L--Pro--OH is then combined at about -5° to 5° C. with a solutioncontaining a substantially equimolar amount of the above dipeptideH--L--(N--Me)Leu--Gly--OEt in the form of its hydrobromic acid additionsalt and an organic base, preferably N-ethylmorpholine in an inertorganic solvent, preferably tetrahydrofuran. The mixture is stirred forabout 30 minutes at about -5° to 5° C. and then about about 20° to 30°C. for about 30 to 50 hours to giveBoc--L--Pro--L--(N--Me)Leu--Gly--OEt, i.e. the intermediate of formula2, wherein R¹ is CH₃, R² is CH₂ CH(CH₃)₂, and R⁷ is OEt. Said last-namedintermediate of formula 2 is identical in all respects to the isomer Bof the intermediate of formula 2 obtained as described above.

The tripeptide derivative of formula 1 in which R¹ is CH₃, R² is CH₂CH(CH₃)₂, R³ is NH₂ and Y is the amino acid residue D--Ala is readilyprepared by condensing an activated ester ofbenzyloxycarbonyl-L-(N-methyl)-leucine, preferably the 1-benzotriazolylester, with D-alanine methyl ester to obtain the dipeptide of formulaZ--L--(N--Me)Leu--D--Ala--OMe. The amino protecting group (Z) of thelatter compound is removed followed by condensation with an activatedester of benzyloxycarbonyl-L-proline, preferably the p-nitrophenylester, to give the tripeptide of formulaZ--L--Pro--L--(N--Me)Leu--D--Ala--OMe. The latter compound is subjectedto the action of ammonia in an inert organic solvent to give thetripeptide amide of formula Z--L--Pro--L--(N--Me)Leu--D--Ala--NH₂. Theamino protecting group (Z) of the latter compound is removed to obtainthe corresponding tripeptide derivative of formula 1Z--L--Pro--L--(N--Me)Leu--D--Ala--NH₂.

In a preferred embodiment of the preparation of the latter tripeptidederivative of formula 1, substantially equimolar amounts ofZ--L--(N--Me)Leu--OH, described by J. R. Coggins, supra, andH--D--Ala--OMe in an inert organic solvent, preferablydimethylformamide, are combined at about 0° to 10° C. with 0.2 to 1.0molar equivalents of 1-hydroxybenzotriazole and a substantiallyequimolar amount of an organic base, preferably N-ethylmorpholine. Asolution of substantially equimolar amounts of dicyclohexylcarbodiimidein an inert organic solvent, preferably tetrahydrofuran is slowly added.After completion of addition the mixture is stirred at about 0° to 10°C. for about one hour and at 20° to 30° C. for another hour. Afterconventional purification the dipeptide of formulaZ--L--(N--Me)Leu--D--Ala--OMe is obtained.

Thereafter the amino protecting group (Z) of the latter dipeptide isremoved, preferably by hydrogenation in the presence of a noble metalcatalyst, preferably 5% palladium on charcoal, in an inert solvent,preferably acetic acid containing a substantially equimolar amount of amineral acid, preferably hydrochloric acid. Removal of the catalyst andevaporation of the solvent gives the dipeptide of formulaH--L--(N--Me)Leu--D--Ala--OMe in the form of its hydrochloric acidaddition salt. The latter compound is condensed with an activated esterof Z--L--Pro--OH. A practical and convenient method for thiscondensation comprises containing substantially equimolar amounts of thelatter dipeptide acid addition salt, 1-hydroxybenzotriazole,benzyloxycarbonyl-L-proline p-nitrophenyl ester and N-ethylmorpholine inan inert organic solvent, preferably dimethylformamide at a temperatureof about 0° to 10° C. The mixture is stirred at 0° to 10° C. for about 2to 4 days. After evaporation of the solvent the residue is dissolved ina substantially water immiscible organic solvent, preferably ethylacetate, washed, dried and evaporated. The residue is purified,preferably by chromatography on silica gel to obtain the tripeptide offormula Z--L--Pro--L--(N--Me)Leu--D--Ala--OMe.

Said last-named compound is subjected to amidation. Preferred conditionsinclude treating the latter compound with a substantially saturatedsolution of ammonia in an inert organic solvent for example, methanol orethanol, at 0° to 10° C. for 2 to 4 days. The solvent is evaporated andthe residue crystallized to obtain the tripeptide of formulaZ--L--Pro--L--(N--Me)Leu--D--Ala--NH₂.

The amino protecting group (Z) of the latter compound is removed,preferably by hydrogenation in the presence of a noble metal catalyst inthe presence of hydrochloric acid as described immediately above, toobtain the tripeptide derivative H--L--Pro--L--(N--Me)Leu--D--Ala--NH₂,i.e. the compound of formula I wherein R¹ is CH₃, R² is CH₂ CH(CH₃)₂, R³is NH₂ and Y is the amino acid residue D--Ala, in the form of thehydrochloric acid addition salt. The acetic acid addition salt of thelatter tripeptide derivative of formula 1 is obtained preferably bysubjecting said hydrochloric acid addition salt to ion exchangechromatography on a column of carboxymethyl cellulose absorbent(Whatmann CM-23) using ammonium acetate buffer as eluant. If desired theacetic acid addition salt of the latter tripeptide derivative of formula1 is subjected to repeated lyophilization from water to obtain thelatter tripeptide derivative of formula 1 in the form of the free base.

The tripeptide derivative of formula 1 in which R¹ is hydrogen, R² isCH₂ CH(CH₃)₂, R³ is NH(CH₂)₄ NH₂ and Y is the amino acid residue Gly isreadily prepared by subjecting the compound of formulaZ--L--Pro--L--Leu--Gly--OEt to hydrolysis to obtain the correspondingacid of formula Z--L--Pro--L--Leu--Gly--OH. Said last-named acid istranformed to an activated ester and the latter is condensed withmono(t-butoxycarbonyl)-1,4-diaminobutane to give the intermediate offormula 2 Z--L--Pro--L--Leu--Gly--NH(CH₂)₄ NH--Boc. The amino protectinggroups of the latter compound are removed to obtain correspondingtripeptide derivative of formula 1 in which R¹ is hydrogen, R² is CH₂CH(CH₃)₂, R³ is NH(CH₂)₄ NH₂ and Y is the amino acid residue Gly.

In a preferred embodiment of the preparation of the latter tripeptidederivative of formula 1 the starting material, the tripeptide of formulaZ--L--Pro--L--Leu--Gly--OEt, described by W. D. Cash, J. Org. Chem., 26,2136 (1961) is treated with a hydrolyzing agent to obtain thecorresponding acid of formula Z--L--Pro--L--Leu--Gly--OH. For basichydrolysis a preferred method involves subjecting the tripeptide esterto the action of strong base, for example, sodium or potassiumhydroxide, in the presence of sufficient water to effect hydrolysis ofthe ester. The hydrolysis is performed using a suitable solvent, forexample, methanol or ethanol. The reaction mixture is maintained at atemperature of about 10° to 30° C. for 15 to 30 min. The reactionmixture is then rendered acidic with an acid, for example, hydrochloricacid, sulfuric acid and the like. The precipitate is collected andcrystallized to obtain the corresponding acid of formulaZ--L--Pro--L--Leu--Gly--OH.

The above acid is treated with a reagent for transforming an amino orpeptide acid to the corresponding activated ester followed by thecondensation of the activated ester with a mono protectedamino(lower)alkyl amine. This reaction is effected by reacting thecorresponding acid with 1.1 to 1.5 molar equivalents ofdicyclohexylcarbodiimide and 1.1 to 2.1 molar equivalents of1-hydroxybenzotriazole followed by the addition of approximatelyequimolar equivalents of mono(t-butoxycarbonyl)-1,4-diaminobutanehydrochloride and an organic base, preferably N-ethylmorpholine, in aninert organic solvent, for example, ethyl acetate, dimethylformamide ortetrahydrofuran at a temperature of from about 0° to 30° C., andreaction times of from three to ten hours. The precipitate is removed,the filtrate evaporated and the residue is dissolved in ethyl acetate.The solution is washed, dried, evaporated and the residue purified,preferably by chromatography on silica to obtain the intermediate offormula 2, Z--L--Pro--L--Leu--Gly--NH(CH₂)₄ NH--Boc.

The amino protective groups Z and Boc are removed from said last-namedcompound to obtain the tripeptide derivativeH--L--Pro--L--Leu--Gly--NH(CH₂)₄ NH₂, i.e. the compound of formula 1wherein R¹ is H, R² is CH₂ CH(CH₃)₃, R³ is NH(CH₂)₄ NH₂, and Y is theamino acid residue Gly. The two amino protective groups may be removedsimultaneously, for example, by using a strong acid, i.e. hydrobromicacid in acetic acid or hydrofluoric acid, or preferably the aminoprotective groups may be removed, in a stepwise manner as follows. Theabove amino protected tripeptide is subjected to hydrogenation asdescribed above in the presence of a noble metal catalyst in an inertsolvent, preferably 5% palladium on charcoal in acetic acid, in order toremove the amino protecting group, benzyloxycarbonyl (Z). The mixture isfiltered and the filtrate cooled to about 0° to 10° C. and treated witha moderately strong acid to remove the remaining amino protecting group,t-butoxycarbonyl (Boc). An example of such an acid is anhydrous hydrogenchloride which is either added directly to the above cooled filtrate ora solution of the acid in an inert organic solvent, for example, ethylacetate, or tetrahydrofuran is added. The mixture is stirred at about 0°to 30° C. for about one to four hours and evaporated to obtain saidtripeptide derivative of formula 1 H--L--Pro--L--Leu--Gly--NH(CH₂)₄ NH₂as the hydrochloric acid addition salt. The acetic acid addition salt ofthe latter tripeptide derivative of formula 1 is obtained by subjectingsaid hydrochloric acid addition salt to ion exchange chromatography,preferably on an anion exchange resin (Baker CGA-540) in the acetateform. If desired, said acetic acid addition salt of the lattertripeptide derivative of formula 1 is subjected to repeatedlyophilization from water to obtain the latter tripeptide derivative offormula 1 in the form of the free base.

Finally, it will be apparent to those skilled in the art that equivalentamino or carboxyl protecting groups, equivalent methods of couplingpeptide fragments, and equivalent methods for removal of the protectinggroups, other than those disclosed herein may be used in the embodimentsof this invention without departing from the scope and spirit of theinvention. Such apparent alternations are intended to be included withinthe scope of this invention.

The following formulae and examples illustrate further this invention.

EXAMPLE 1Benzyloxycarbonyl--L--prolyl--DL--(N--dimethylamino)leucyl--glycineEthyl Ester (Z--Pro--N[N(CH₃)₂ ]CH[CH₂ CH(CH₃)₂ ]CO--Gly--OEt)

a. Benzyloxycarbonyl-L-proline (12.45 g, 50 mmoles) in dry methylenechloride (50 ml) is added to a solution at 0° C. of isovaleraldehydeN,N-dimethylhydrazone (7.05 g, 55 mmoles) and ethylisocyanoacetate (6.21g, 55 mmoles) in dry methylene chloride (50 ml). The mixture is stirredat room temperature for 4 days, washed with 5% sodium bicarbonatesolution and saturated sodium chloride solution, dried, filtered and thefiltrate evaporated. The residue is subjected to chromatography onsilical gel using chloroform-methanol (98:2) as eluant. Evaporation ofthe solvent gives the title compound.

b. In the same manner but replacing benzyloxycarbonyl-L-proline witht-butoxycarbonyl-L-proline and replacing isovaleraldehyde.Iadd..[.N,N-dimethylhydrozone.]..Iaddend. .Iadd.N,N-dimethylhydrazone.Iaddend.with methylamino-N-isopentylidene, the two isomers A and B, oft-butoxycarbonyl-L-prolyl-DL-(N-methyl)leucylglycine ethylester.[.(Boc--L--Pro--N(CH₃)CH[CH₂ CH(CH₃)₂ ]CO--Gly--OFt.]..Iadd.(Boc--L--Pro--N(CH₃)CH[CH₂ CH(CH₃)₂ ]CO--Gly--OEt .Iaddend.areobtained; isomer A [α]_(D) ²⁵ =+31.8°(c=1, dimethylformamide), nmr(CDCl₃) δ 1.0 (m, 6H), 1.3 (t, J=7Hz, 3H), 1.4 (s, 9H), 3.1 (s, 3H), 4.2(q, J=7Hz). Isomer B, [α]_(D) ²⁵ =-76.7° (c=1, dimethyl- formamide),mass spectrometry (m/e): 427 (M⁺).

c. In the same manner but replacing benzyloxycarbonyl-L-proline witht-butoxycarbonyl-L-proline and replacing isovaleraldehydeN,N-dimethylhydrazone with a mixture of formaldehyde and isobutylamine,t-butoxycarbonyl-L-prolyl-(N-isobutyl)-glycyl-glycine .[.glycine.].ethyl ester.[.(Boc--L--Pro--N[CH₂ CH(CH₃)₂ ]CH₂ CO--Gly--OE)dimethylformamide),.]. .Iadd.(Boc--L--Pro--N[CH₂ CH(CH₃)₂ ]CH₂CO--Gly--OEt), is .Iaddend.obtained: nmr (CDCl₃) δ0.9 (6H), 1.25 (3H),1.40 (9H), 2.0 (5H), 3-5 (11H), 7.8 (1H).

d. In the same manner but replacing isovaleraldehydeN,N-dimethylhydrazone with a mixture of 1,1-diethylhydrazine andacetaldehyde,benzyloxycarbonyl-L-prolyl-DL-(N-diethylamino)-alanyl-glycine ethylester (Z--L--Pro--N[N(C₂ H₅)₂ ]CH(CH₃)CO--Gly--OE is obtained.

e. In the same manner but replacing isovaleraldehydeN,N-dimethylhydrazone with a mixture of 1,1-di(n-propyl)hydrazine andisobutyraldehyde,benzyloxycarbonyl-L-prolyl-DL-[N-di-(n-propyl)amino]valyl-glycine ethylester (Z--L--Pro--N[N(CH₂ CH₂ CH₃)₂ ]CH[CH(CH₃)₂ ]CO--Gly--OEt isobtained.

f. In the same manner but replacing isovaleraldehydeN,N-dimethylhydrazone with a mixture of ethylamine and isobutyraldehyde,benzyloxycarbonyl--L--prolyl--DL--(N-ethyl)valyl-glycine ethyl ester(Z--L--Pro--N(C₂ H₅)CH[CH(CH₃)₂ ]CO--Gly--OEt) is obtained.

EXAMPLE 2 Benzyloxycarbonyl--L--prolyl--DL--(N--dimethylamino)leucyl--glycinamide Z--Pro--N[N(CH₃)₂ ]CH[CH₂ CH(CH₃)₂ ]CO--Gly--NH₂

a. The intermediate, benzyloxycarbonyl-L-prolyl-DL-(N-dimethylamino)leucyl-glycine ethyl ester, (8.33 g, 17 mmoles, described in Example 1(a) is dissolved at 0° C. in a saturated solution of anhydrous ammoniain dry methanol (150 ml) and allowed to stand at 0° C. for 3 days. Themixture is evaporated under reduced pressure and the residue issubjected to chromatography on silica gel using chloroform-methanol(95:5) as eluant. The two isomers A and B, of the title compound areeluted separately.

Isomer A, nmr (CDCl₃) δ0.97 (d, J ═6Hz), 2.28 (s, 6H), 5.00 (s, 2H),7.29 (s, 5H).

Isomer B, nmr (CDCl₃) δ0.95(t, J ═6Hz, 6H), 2.25 (s, 6H), 5.10 (s, 2H),7.29 (s, 5H).

b. In the same manner but replacingbenzyloxy-carbonyl-L-prolyl-DL-(N-dimethylamino) leucyl-glycine ethylester with an equivalent amount of isomer A of.[.-butoxy-carbonyl.]..Iadd.-t-butoxycarbonyl.Iaddend.-L-prolyl-DL-(N-methyl)leucyl-glycine ethyl ester (described in Example 1 (b), the isomer A of.Iadd.t.Iaddend.-butoxycarbonyl-L-prolyl-DL-(N-methyl)leucyl-glycinamide(Boc--L--Pro--N(CH₃)CH[CH₂ CH(.[.CH₃₁₁ .]..Iadd.CH₃).Iaddend.₂]CO--Gly--NH₂) is obtained, m.p. 130°-132° C., [α]25/D═+50.2° (c═2,dimethylformamide).

In the same manner but replacing benzyloxycarbonyl-L-proyl-DL-(N-dimethylamino) leucyl-glycine ethyl ester with anequivalent amount of isomer B of.Iadd.t.Iaddend.-butoxycarbonyl-L-.[.proly.]..Iadd.prolyl.Iaddend.-DL-(N-methyl)leucyl-glycine ethyl ester (described in Example 1(b), then the isomer Bof .Iadd.t.Iaddend.-butoxycarbonyl-L-prolyl-DL-(N-methyl).[.leuceylglycinamide.]. .Iadd.leucyl-glycinimide.Iaddend.(Boc--L--Pro--N(CH₃)CH[CH₂ -CH(CH₃)₂ ]Gly--NH₂ is obtained,mass spectrometry (m/e): 398 (m⁺).

c. In the same manner but replacingbenzyloxycarbonyl-L-propyl-DI-(N-.[.diemthylamino.]..Iadd.dimethylamino.Iaddend.)leucyl-glycine ethyl ester with anequivalent amount of -butoxycarbonyl-L-prolyl-(N-isobutyl)-glycyl-glycine ethyl ester (described in Example 1 (c),.Iadd.t.Iaddend.-butoxycarbonyl-L-prolyl-(N-isobutyl)glycly-glycinamide(Boc--L--Pro--N[CH₂ CH(CH₃)₂ ]CH₂ CO--Gly--NH₂ ]is obtained: m.p.92°-95° C., nmr (CDCl₃) δ0.9 and 1.03 (doublets, J ═2.5 Hz, 6H), 1.4 (s,9H).

EXAMPLE 3 L-Prolyl-DL-(N-dimethylamino)leucyl-glycinamide hydrochloride1; R¹ ═N(CH₃)₂, R² ═CH₂ CH(CH₃)₂, R³ ═NH₂, Y═Gly; H--L--Pro--N[N(CH₃)₂]CH[CH₂ CH(CH₃)₂ ]CO--Gly--NH₂, HCl

a. A mixture ofbenzyloxycarbonyl-L-prolyl-DL-(N-dimethylamino)leucyl-glycinamide [1.595g, 3.46 mmoles, isomer A, described in Example 2(a)]and 5% palladium oncharcoal catalyst (0.207 g) in methanol is stirred under an atmosphereof hydrogen for 17 hr with the hydrogenation vessel connected to a flaskcontaining a stirred sodium hydroxide solution (4n, 100 ml). Thecatalyst is removed by filtration. Methanolic hydrochloric acid (0.94N,3.7 ml, 3.47 mmoles) is added to the filtrate and the latter isevaporated under reduced pressure. The residue is decolorized withactive charcoal in anhydrous methanol, filtered and the filtrateevaporated under reduced pressure. The residue is triturated withdiethyl ether, ethyl acetate and diethyl ether and dried under reducedpressure over phosphorus pentoxide to give isomer A of the titlecompound; [α]_(D) ²⁵ ═-43.9° (c ═2, dimethylformamide), nmr (DMSO-d₆)δ0.9 (d, J ═5Hz, 6 H), 2.57 (s, 6H).

In the same manner but replacing isomer A with an equivalent amount ofthe corresponding isomer B ofbenzloxycarbonyl-L-prolyl-DL-(N-dimethylamino)leucyl-glycinamide[described in Example 2(a)], the corresponding isomer B of the titlecompound is obtained: [α]_(D) ²⁵ ═-33° (c ═2, dimethylformamide).

b. A solution of anhydrous hydrogen chloride in dry ethyl acetate (1.5N,55 ml, 82.5 mmoles) is added dropwise over 40 min. to an ice bath cooledsuspension of isomer A oft-butoxycarbonyl-L-prolyl-(N-Methyl)-leucyl-glycinamide [6.56 g, 16.5mmoles, described in Example 2(b)]. The mixture is stirred at ice bathtemperature for 30 min. and at room temperature for 17 hr. The solventis decanted, the solid is triturated with dry ethyl acetate and thesolvent decanted. The residue is dissolved in anhydrous methanol, thesolution filtered and the filtrate evaporated under reduced pressure.The residue is triturated with ethyl acetate-petroleum ether (1:1),diethyl ether-petroleum ether (1:1) and diethyl ether. The residue isdried under reduced pressure over phosphorus pentoxide and potassiumhydroxide to give isomer A of L-prolyl-DL-(N-methyl)leucyl-glycinamidehydrochloride [(1; R¹ ═CH₃, R² ═CH₂ CH(CH₃)₂, R³ ═NH₂, Y═Gly;H--L--Pro--N(CH₃)CH[CH₂ CH(CH₃)₂ ]CO--Gly--NH₂ ] , mass spectrometry(m/e): 298 (M⁺).

In the same manner but replacing isomer A oft-butoxycarbonyl-L-propyl-DL-(N-methyl)leucyl-glycinamide with anequivalent amount of isomer B oft-butoxycarbonyl-L-prolyl-DL-(N-methyl)leucylglycinamide (described inExample 2(b)), isomer B of L-prolyl-DI-(N-methyl)leucyl-glycinamidehydrochloride [1; L R¹ ═CH₃, R² ═CH₂ CH(CH₃)₂, R³ ═NH₂, Y═Gly;H--L--Pro--N(CH₃)CH[CH₂ CH(CH₃)₂ ]CO--Gly--NH₂ ]is obtained; massspectrometry (m/e): 298 (M⁺). c. In the same manner as described inExample 3(b) but replacing isomer A oft-butoxycarbonyl-L-prolyl-DL-(N-methyl)leucylglycinamide with anequivalent amount oft-butoxycarbonyl-L-prolyl(N-isobutyl)glycl-glycinamide,L-prolyl-(N-isobutyl)glycylglycinamide hydrochloride [1; R¹ ═CH₂CH(CH₃)₂, R² ═H, Y ═Gly, R³ ═NH₂ ; H--L--Pro--N[CH₂ CH(CH₃)₂ ]CH₂CO--Gly--NH₂ ] is obtained; mass spectrometry (m/e): 284 (M⁺).

EXAMPLE 4L-Prolyl-DL-(N-dimethylamino)leucyl-glycine-4-amino-n-butyl-amidedihydrochloride; 1; R¹ ═ N(CH₃)₂, R² ═CH₂ CH(CH₃)₂, R³ ═ NH(CH₂)₄ NH₂,Y═ Gly; (H--L--Pro--N[N(CH₃)₂ ]CH[CH₂ CH(CH₃)₂ ]CO--Gly--NH(CH₂)₄ NH₂ .2 HCl

a. A solution ofbenzyloxycarbonyl-L-prolyl-DL-(N-dimethylamino)leucyl-glycine ethylester (4.45 g, 9.07 mmoles, described in Example 1(a)) and IN sodiumhydroxide (12.25 ml) in methanol (23 ml) is stirred at room temperaturefor 20 hr. Saturated sodium chloride solution (50 ml) is added and theprecipitate removed by filtration. The filtrate is cooled to 0° C. andacidified with IN hydrochloric acid (13.3 ml). The mixture is extractedwith chloroform. The organic extract is washed with water to neutral,dried over magnesium sulfate and evaporated under reduced pressure togive benzyloxycarbonyl-L-prolyl-DL-(N-dimethylamino)-leucyl-glycine; nmr(CDCl₃) δ0.93 (6H), 2.53 (6H), 8.6 (1H).

b. N,N'-Carbonyldiimidazole (1.19 g, 7.5 mmoles) is added to a stirredsolution at -15° C. ofbenzyloxycarbonyl-L-prolyl-DL-(N-dimethylamino)leucyl-glycine [3.477 g,7.5 mmoles, described in Example 4(a)]in dry dimethylformamide (14 ml)maintaining a dry atmosphere. The mixture is stirred at -15° C. for 30min. A solution of mono-(t-butoxycarbonyl)-1,4-diaminobutanehydrochloride [1.685 g, 7.5 mmoles, prepared as described by R. Geiger,Annalen, 750, 165 (1971)]and triethylamine (1.25 ml) in drydimethylformamide (6 ml) is added. The mixture is stirred at roomtemperature for 20 hr. and evaporated. The residue is taken up in ethylacetate (200 ml) and washed with 10% sodium bicarbonate solution, 20%sodium chloride solution, 10% citric acid solution, and 20% sodiumchloride solution. The organic phase is dried over magnesium sulfate andevaporated. The residue is subjected to chromatography on silica gelusing chloroform-methanol-pyridine(95:5:1) to elute separately the twoisomers, A and B, ofbenzyloxycarbonyl-L-prolyl-DL-(N-dimethylamino)leucyl-glycine-4-t-butoxycarbonyl-amino-n-butylamide:

Isomer A; nmr (CDCl₃) δ0.99 (d, J═6Hz, 6H), 1.42 (s, 9H), 2.65 (6H), 5(s, 2H), 7.25 (5H).

Isomer B, nmr (CDCl₃) δ0.95 (t, J═6Hz, 6H), 1.43 (s, 9H), 5.10 (s, 2H),7.30 (s, 5H).

c. A mixture of isomer A ofbenzyloxycarbonyl-L-prolyl-DL-(N-dimethylamino)leucylglycine-4-t-butoxycarbonylamino-n-butyl-amide [2.26 g, 3.58 mmole,described in Example 4(b)]and 5% palladium on charcoal catalyst (0.250g) in acetic acid (50 ml) is stirred under an atmosphere of hydrogen for21 hr with the hydrogenation vessel connected to a flask containing astirred solution of sodium hydroxide (4N, 100 ml). The catalyst isremoved by filtration, the filtrate cooled in ice-water and a solutionof hydrogen chloride in dry ethyl acetate (1.6 N, 12 ml) is addeddropwise. The mixture is stirred at room temperature for two hours withexclusion of moisture. The solvent is evaporated under reduced pressureand traces of acetic acid are removed by azeotropic evaporation with drybenzene. The residue is subjected to chromatography on a column of across-linked dextran absorbent (Sephadex LH-20) using methanol. Theeluant is decolorized with active charcoal, filtered and evaporated. Theresidue is triturated with anhydrous diethyl ether and dried underreduced pressure over phosphorus pentoxide to give isomer A of the titlecompound: [α]_(D) ²⁵ ═-43.6° (c═2, dimethylformamide);

Analysis for C₁₉ H₃₈ N₆ O₃ 2 HCl; Calc'd: C, 47.70; H, 8.52; N, 17.60;Cl, 14.86; Found: C, 47.10; H, 8.55; N, 17.55; Cl, 15.24.

In the same maner manner replacing isomer A ofbenzyloxycarbonyl-L-proplyl-DL-(N-dimethylamino)-leucyl-glycine-4-t-butoxycarbonylamino-n-butyl-amidewith the corresponding isomer B [described in Example 4(b)], thecorresponding isomer B of the title compound is obtained: [α]_(D) ²⁵-18.6° (c═2, dimethylformamide):

Analysis for C₁₉ H₃₆ N₆ O₃ 2 HCl H₂ O: Calc'd: C, 46.62; H, 8.64; N,17.20; Found: C, 46.88; H, 8.50; N, 17.51.

d. In the same manner but replacingbenzyloxycarbonyl-L-prolyl-DL-(N-.[.diemethylamino.]..Iadd.dimethylamino.Iaddend.)leucyl-glycine ethyl ester with an equivalent amount ofbenzyloxycarbonyl-L-prolyl-DL-(N-diethylamino)-alanyl-glycine ethylester [described in Example 1(d)] and replacingmono-(t-butoxycarbonyl)-1,4-diaminobutane .[.hyrochloride.]..Iadd.hydrochloride .Iaddend.with an equivalent amount of diethylamine,L-prolyl-DL-(N-diethylamino)-alanyl-glycine diethylamide hydrochloride[1; R¹ ═N(CH₂ CH₃)₂, R² ═CH₃, R³ ═N(CH₂ CH₃)₂, Y ═Gly] is obtained.

e. In the same manner but replacingbenzyloxycarbonyl-L-prolyl-DL-(N-dimethylamino)leucyl-glycine ethylester with an equivalent amount ofbenzyloxycarbonyl-L-prolyl-DL-[N-di(n-propyl)amino]valyl-glycine ethylester [described in Example 1(e)] and replacingmono-(t-butoxycarbonyl)-1,4-diaminobutane hydrochloride with anequivalent amount of n-propylamine,L-prolyl-DL-[N-di(n-propyl)amino]valyl-glycine N-propylamidehydrochloride [1; R¹ ═N(CH₂ CH₂ CH₃)₂, R² ═ CH(CH₃)₂, R³ ═NH(CH₂ CH₂CH₃), Y ═ Gly; H--L--Pro--N[N(CH₂ CH₂ CH₃)₂ ]CO-Gly-NH(CH₂ CH₂ CH₃)] isobtained.

f. In the same manner but replacingbenzyloxycarbonyl-L-prolyl-DL-(N-dimethylamino)leucyl-glycine ethylester with an equivalent amount ofbenzyloxycarbonyl-L-prolyl-DL-(N-ethyl)valylglycine ethyl ester[described in Example 1(f)] and replacingmono(t-butoxycarbonyl)-1,4-diaminobutane hydrochloride with anequivalent amount of methylamine, L-prolyl-DL-(N-ethyl)valylglycinemethylamide hydrochloride [1; R¹ ═CH₂ CH₃, R² ═CH(CH₃)₂, R³ ═ NHCH₃, Y═Gly; H--L--Pro--N(CH₂ CH₃)CH[CH(CH₃)₂ ]CO--Gly--NHCH₃ ] is obtained.

EXAMPLE 5 Alternative synthesis oft-butoxycarbonyl-L-prolyl-L-(N-methyl)leucylglycine ethyl ester(Boc--L--Pro--N(CH₃)CH[CH₂ CH(CH₃)₂ ]CO--Gly--OEt (isomer B, previouslydescribed in Example 1(b))

A solution of Z-L-(N-Me)Leu-OH [4.2 g, 15 mmoles, prepared as describedby J. R. Coggins and N. Leo Benoiton, Can. J. Chem., 49, 1968 (1972)],2,4,5-trichlorophenol (2.96 g, 15 mmoles) and dicyclohexylcarbodiimide(3.09 g, 15 mmoles) in dry methylene chloride (21 ml) is stirred at -15°C. for one hr and then at room temperature for 2 hr. The.[.pecipitate.]. .Iadd.precipitate .Iaddend.is removed by filtration andthe filtrate is evaporated to give Z--L--(N--Me)-Leu--Otcp.

A solution of Z--L--(N--Me)Leu--OTcp (15 mmoles, described above) indimethylformamide (10.5 ml) is added to a solution of 0° C. of glycineethyl ester hydrochloride (2.09 g, 15 mmoles) and N-ethylmorpholine(1.92 l ml, 15 mmoles) in dimethylformamide (35 ml) and the mixture isstirred at 0° C. for 30 min and then at room temperature for 20 hr. Thesolvent is removed by evaporation and the residue is subjected tochromatography on silica gel using chloroform-ethyl acetate (85:15) aseluant. Evaporation of the eluates gives Z--L--(N--Me)Leu--Gly--OEt, nmr(CDCl₃) δ 0.93 (6H), 1.27 (3H), 2.85 (3H), 3.97 (2H), 4.20 (2H), 5.17(2H), 7.34 (5H).

A solution of hydrobromic acid in acetic acid (30-32%, 4.9 ml, 24mmoles) is added to a solution of Z--L--(N--Me)Leu--Gly--OEt (2.9 g,7.96 mmoles, described above) in acetic acid (4.7 and the resultingmixture is stirred at room temperature for 4.5 hr. The solvent isremoved by evaporation under reduced pressure and the esidue residuesubjected to repeated azeotropic distillation with benzene-methanol. Theresulting residue is dried under reduced pressure over potassiumhydroxide to give [H--L--(N--Me)Leu--Gly--OEt]HBr.

A solution of Boc-- L--Pro--OH (0.645 g, 3 mmoles),1-hydroxybenzotriazole (0.810 g, 6 mmoles) and dicyclohexylcarbodiimide(0.680, 3.3 mmoles) in dry tetrahydrofuran (15 ml) is stirred at -5° C.for one hr and then at 25° C. for one hr. The mixture is cooled to 0° C.and treated with a solution at 0° C. of [H--L--(N--Me)Leu--Gly--OEt]Hbr(3 mmoles, described above) and N-ethylmorpholine (0.384 ml, 3 mmoles)in dry tetrahydrofuran (14 ml). The mixture is stirred at 0° C. for 30min and then at room temperature for 40 hr. The .[.pecipitate.]..Iadd.precipitate .Iaddend.is removed by filtration and the filtrate isevaporated under reduced pressure. The residue is dissolved in ethylacetate (100 ml). The solution is washed with ice-cold IN citric acid,water, 5% sodium bicarbonate solution and saturated sodium chloridesolution. The organic phase is dried over magnesium sulfate andevaporated. the residue is subjected to chromatography on silica gelusing chloroform-ethyl acetate-pyridine (50:50:01) as eluant to give thetitle compound, t-butoxycarbonyl-L-propyl-L-(N-methyl)-leucylglycineethyl ester; [α]D²⁵ ═-73.1° (c═1, dimethylformamide). The title compoundobtained by the above method is identical to isomer B oft-butoxycarbonyl-L-prolyl-DL-(N-methyl)leucyl-glycine ethyl ester asobtained by the method described in Example 1(b).

EXAMPLE 6 L-prolyl-L-(N-methyl)leucyl-D-alaninamide(H-Pro-L-(N-Me)Leu-D-Ala-NH₂) [1; R¹ ═CH₃, R² ═CH₂ CH(CH₃)₂, R³ ═NH₂, Y═D-Ala]

.[.Benxyloxycarbonyl.]..Iadd.Benzyloxycarbonyl.Iaddend.-L-(N-methyl)leucine (14.9 g, 50mmoles), D-alanine methyl ester (7.0 g, 50 mmoles) and1hydroxybenzotriazole (13.5 g, 10 mmoles) are dissolved in a mixture ofdry tetrahydrofuran (300 ml) and dimethylformamide (70 ml). The solutionis cooled to 0° C. and N-ethylmorpholine (6.42 ml; 50 mmoles) is addedfollowed by dropwise addition of a solution of dicyclohexylcarbodiimide(10.3 g; 50 mmoles) in dry tetrahydrofuran (80 ml). The reaction mixtureis stirred at 0° C. for 1 hour, at room temperature for a further hour,filtered and the solvents removed .[.uner.]. .Iadd.under.Iaddend.reduced pressure. The residue is taken in ethyl acetate andextracted with water and saturated sodium chloride solution. The residueleft after drying and evaporation of the ethyl acetate layers issubjected to chromatography on silica gel (1 kg; CHCl₃ containing 2%MeOH). Evaporation of the eluants gives Z--L--(N--Me)Leu--D--Ala--OMe;nmr (CDCl₃) δ 2.86 (3H, s), 3.74 (3H, s), 5.25 (; 2H, s), 7.42 (5H, s).

A mixture of benzyloxycarbonyl-L-(N-methyl)leucyl-D-alanine methyl ester(10.5 g, 28.8 g, described above) and 5% palladium on charcoal (1.0 g)in acetic acid (120 ml) and hydrochloric acid (2N, 14.4 ml, 28.8 mmoles)is stirred under an atmosphere of hydrogen for 24 hr. The catalyst isremoved by filtration and the filtrate evaporated to give a residue ofH--L--(N--Me)Leu--D--Ala--OMe. HCl. A solution of the latter compound(28.8 mmoles), 1-hydroxybenzotriazole (3.9 g, 28.8 mmoles),benzyloxycarbonyl-L-proline p-nitrophenyl ester (10.7 g, 28.8 mmoles)and N-ethylmorpholine (3.7 ml, 28.8 mmoles) in dimethyl-formamide (70ml) is stirred at 0° C. for three days. After evaporation of thesolvents under reduced pressure the residue is dissolved in ethylacetate and washed with water, saturated sodium bicarbonate solution,water and saturated sodium chloride solution. The organic phase is driedover magnesium sulfate and evaporated. The residue is subjected tochromatography on silica gel using chloroform-methanol (98:2). Theeluants are evaporated to givebenzyloxycarbonyl-L-prolyl-L-(N-methyl)leucyl-D-alanine methyl ester(Z--L--Pro--L--(N--Me)Leu--D--Ala--OMe); nmr (CDCl₃); δ 0.95 (d, 6H),2.87 and 3.0 (two s, 3H), 3.72 (s, 3H), 7.4 (s, 5H).

The latter compound (1.7 g, 3.68 mmoles) is dissolved at 0° C. inmethanol saturated with ammonia (85 ml) and allowed to stand at 0° for 3days. The solvent is removed by evaporation and the residue iscrystallized from isopropyl ether-acetone to givebenzyloxycarbonyl-L-prolyl-L-(N-methyl)leucyl-D-alaninamide(Z-L-pro-L-(N-Me)Leu-D-Ala-NH₂); m.p. 148°-150° C.,

Analysis for C₂₃ H₃₄ N₄ O₅ : Calc'd: C, 61.86; H, 7.67; N, 12,55%;Found: C, 61.67; H, 7.82, N, 12.66%.

A mixture of Z--L--Pro--L--(N--Me)--Leu--D--Ala--NH₂ (1.29 g, 2.9mmoles, described above) and 5% palladium on charcoal (0.13 g) in aceticacid (20 ml) and hydrochloric acid (IN, 2.9 ml) is stirred under andatmosphere of hydrogen for 20 hr. The catalyst is removed by filtrationand the filtrate is lyophilized to give the title compound as thehydrochloric acid addition salt. The residue is subjected to ionexchange chromatography on a column of carboxymethyl cellulose (WhatmannCM-23) using 0.04N aqueous ammonium acetate. The eluant is lyophilizedto give the title compound as the acetic acid addition salt, [α]_(D) ²⁵═-77.6° (c═1, 1% acetic acid).

Repeated lyophilization of the latter compound from water gives thetitle compound as the free base, nmr (CDCl₃) δ 0.90 (s, 6H), 1.22 (d, J═ 7Hz, 3H), 1.45-2.16 (m,H), 2.82 and 2.90 (singlets, 3H), 3.9-5.2 (m,3H).

In the same manner, by using benzyloxycarbonyl-D-(N-methyl)leucine asthe starting material instead of the L-enantiomer described above,L-prolyl-D-(N-methyl)leucyl-D-alaninamide(H--Pro--D--(N--Me)Leu--D--Ala--NH₂) is obtained, amino acid analysis:Pro 0.88, Ala 1.00.

EXAMPLE 7

L-prolyl-L-leucyl-glycine-4-amino-n-butyl-amide [1; R¹ ═H, R² ═CH₂CH(CH₃)₂, R³ ═NH(CH₂)₄ NH₂, Y═ Gly] (H--L--Pro--L-Leu--Gly--NH(CH₂)₄NH₂)

IN sodium hydroxide (3.02 ml) is added dropwise to a stirred suspensionat 0° C. of benzyloxycarbonyl-L-prolyl-L-leucyl-glycine ethyl ester [1.0g, 2.24 mmoles, prepared as described by W. O. Cash, J. Org. Chem., 26,2136 (1961)]in methanol (5.6 ml) and the solution is stirred at 25° C.for 20 min. The solution is diluted with saturated sodium chloride (23ml), cooled to 0° C. and acidified with IN hydrochloric acid (3.3 ml).The mixture is stirred at 0° C. for 20 min. The solid is collected,washed with cold water, dried .[.uner.]. .Iadd.under .Iaddend.reducedpressure over phosphorus pentoxide, and crystallized from methanol-waterto give Z--L--Pro--L--Lue--Gly--OH; m.p. 162°-165° C., [α]_(D) ²⁵═-55.8° (C═2, dimethylformamide).

A mixture of the latter compound (9.48 g, 22.6 mmoles)mono-(t-butoxycarbonyl)-1,4-diaminobutane hydrochloride (5.06 g, 22.6mmoles), N-ethylmorpholine (28.9 ml), 1-hydroxybenzotriazle (6.1 g, 45mmoles) and dicyclohexylcarbodiimide (4.98 g, 24.85 mmoles) indimethylformamide (225 ml) is stirred at 0° C. for one hour and then at25° C. for 4 hr. The .[.pecipitate.]. .Iadd.precipitate .Iaddend.isremoved by filtration and the solvent evaporated. The residue isdissolved in ethyl acetate, the precipitate is removed, and the filtrateis washed with 10% sodium bicarbonate solution, water, 10% citric acidsolution and water. The organic phase is dried over magnesium sulfateand the solvent evaporated. The residue is subjected to chromatographyon silica gel using chloroform-methanol-pyridine (98:2:1) as the eluantfollowed by evaporation of the eluant to giveZ--L--Pro--L--Leu--Gly--NH(CH₂)₄ NH--Box, nmr (CDCl₃): δ0.92 (6H), 1.42(9 H), 5.16 (2H), 7.36 (5H).

A mixture of the latter compound (7.4 g, 12.55 mmoles) and 5% palladiumon charcoal (0.505 g) in acetic acid (50 ml) is stirred under anatmosphere of hydrogen for 5 hr with the hydrogenation vessel connectedto a flask containing a stirred solution of sodium hydroxide 4N, 250ml). The mixture is filtered and the filtrate cooled in an ice bath. Asolution of hydrogen chloride in dry ethyl acetate (4.6N, 16 ml) isadded dropwise and the mixture is stirred at 10° C. for 30 min and thenat room temperature for 3 hr. The solvent is evaporated and the residueis dissolved in benzene and the solvent is again evaporated. The residueis dissolved in methanol, active charcoal is added, filtered and thesolvent evaporated from the filtrate to give the title compound as thehydrochloric acid addition salt. The latter compound is dissolved in 0.1N hydrochloric acid and subjected to ion exchange chromatography on ananinon exchange resin (Baker CGA-540) in the acetate form. The eluantsare evaporated, the residue triturated with diethyl ether and petroleumether and dried to give the title compound as the acetic acid additionsalt;

Anal. for C₁₇ H₃₃ N₅ O₃. 2CH₃ CO₂ H. 1/2 H₂ O (484.6): Calc'd: C, 52.04;H, 8.73; N, 14.45; CH₃ CO₂ H, 24.78%, Found: C, 52.13; H, 8.70; N,14.35; CH₃ CO₂ H, 23.2%.

We claim:
 1. A compound .[.of formula 1.]. .[. .]. .[.in which R¹ ishydrogen, lower alkyl or NR⁴ R⁵ wherein R⁴ and R⁵ each are lower alkyl,R² is hydrogen or lower alkyl, R³ is amino, lower alkylamino,di(lower)alkylamino or amino(lower)alkylamino, and Y is one of the aminoacid residues Gly or D--Ala with the proviso that when R¹ is NR⁴ R⁵ islower alkylamino, di(lower)alkylamino or amino(lower)alkylamino and withthe further proviso that when R¹ is hydrogen, R² is CH₂ CH(CH₃)₂, R³ isamino then Y is D--Ala..]. .Iadd.selected from the group consisting ofL-prolyl-D-(N-methyl)leucyl-glycinamide,L-prolyl-L-(N-methyl)leucyl-glycinamide,L-propyl-(N-isobutyl)glycyl-glycinamide,L-prolyl-DL-(N-dimethylamino)leucyl-glycine-4-amino-n-butylamide,L-prolyl-L-(N-methyl)leucyl-D-alaninamide,L-prolyl-N-(D-methyl)-leucyl-D-alaninamide and L
 2. A pharmaceuticallyacceptable acid addition salt of the compound .[.of
 3. The correspondingamino protected derivative of the compound .[.of formula 1.]. as claimedin claim 1 wherein the protecting group is selected from the groupconsisting of benzyloxycarbonyl, t-butoxycarbonyl,α,α-dimethyl-3,4,-dimethoxybenzyloxycarbonyl, triphenylmethyl 7.L-Prolyl-DL-(N-dimethylamino)leucyl .Iadd.-glycine-4-amino-.Iaddend.n-butyl-amide, as claimed in claim 1 having a rotation of 8.L-Prolyl-DL-(N-dimethylamino)leucyl-glycine-4-amino- n-butyl-amide, asclaimed in claim 1 having a rotation of _(D) ²⁵ ═18.6° 11.L-Prolyl-L-leucyl-glycine-4-amino-n-butyl-amide as claimed in claim 1.12. A process for preparing a compound of formula 1 ##STR3## in which R¹is lower alkyl or NR⁴ R⁵ wherein R⁴ and R⁵ each are lower alkyl; R² ishydrogen or lower alkyl; R³ is amino, lower alkylamino,di(lower)alkylamino or amino(lower)alkylamino and Y is the amino acidresidue Gly with the proviso that when R¹ is NR⁴ R⁵, R³ is loweralkylamino di(lower)alkylamino or amino(lower)alkylamino whichcomprises:condensation of an enamine or hydrazone of formula 3, R¹N═CHR² (3), in which R¹ and R² are as defined herein with an amino acidof formula R⁶ --L--Pro--OH in which R⁶ is an amino protecting groupselected from the group consisting of benzyloxycarbonyl,t-butoxycarbonyl, α,α-dimethyl-3,4-dimethoxy benzyloxycarboxyl,triphenylmethyl and benzyl in the presence of an isonitrile of theformula CNCH₂ COR⁷ in which R⁷ is lower alkoxy, to obtain thecorresponding intermediate of formula 2

    R.sup.6 --L--Pro--N(R.sup.1)CH(R.sup.2)CO--Gly--R.sup.7    ( 2)

in which R¹, R², R⁶ and R⁷ are as defined herein, followed by subjectingthe last-named compound to amidation to give the corresponding amide orsubstituted amide and removal of the protective group(s) to obtain thecorresponding peptide derivative of formula 1 in which R¹, R², R³ and Yare as defined herein.
 13. A process as claimed in claim 12 in which thetransformation of the intermediate of formula 2

    R.sup.6 --L--Pro--N(R.sup.1)CH(R.sup.2)CO--Gly--R.sup.7    ( 2)

in which R¹, R², R⁶ and R⁷ are as defined therein is carried out bytreating said intermediate with ammonia to obtain the correspondingamide, followed by removel of the protective group R⁶ selected from thegroup consisting of benzyloxycarbonyl, t-butoxycarbonyl,α,α-dimethyl-3,4-dimethoxy benzyloxycarbonyl, triphenylmethyl and benzylto obtain the corresponding peptide derivative of formula 1 in which R¹and R² are as defined therein, R³ is amino and Y is the amino acidresidue Gly.
 14. A process as claimed in claim 13 in which thetransformation of the intermediate of formula 2

    R.sup.6 --L--Pro--N(R.sup.1)CH(R.sup.2)CO--Gly--R.sup.7    ( 2)

in which R¹, R², R⁶ and R⁷ are as defined therein is carried out bytreating said intermediate with a hydrolyzing agent to obtain thecorresponding acid, treating said acid with an activating agent to givethe corresponding activated ester selected from the group consisting of2,4,5trichlorophenyl, pentachlorophenyl, p-nitrophenyl, and1-benzotriazolyl, and N,N¹ -carbonyldiimidazolide and .[.o-acylura.]..Iadd.O-acylurea .Iaddend.of dicyclohexylcarboxdiimide condensing saidactivated compound with a lower alkylamine, di(lower)alkylamine or monoprotected amino(lower)alkylamine in which the protecting group isselected from the group consisting of benzyloxycarbonyl,t-butoxycarbonyl,α,α-dimethyl 3,4-.[.dimethoxybenzyloxycarbonyol.]..Iadd.dimethoxybenzyloxycarbonyl, .Iaddend.triphenylmethyl and benzyl toobtain the corresponding substituted amide and removing the protectivegroup(s) from said last-named compound to obtain the correspondingpeptide derivative of formula 1 in which R¹ and R² are as definedherein, R³ is lower alkylamino, di(lower)alkylamino oramino(lower)alkylamino and Y is the amino acid residue Gly.
 15. Aprocess for preparing a compound of formula 1 ##STR4## in which R¹ isCH₃, R² is CH₂ CH(CH₃)₂, R³ is NH₂ and Y is the amino acid residueD--Ala which comprises:reacting an activated ester selected from thegroup consisting of 2,4,5, trichlorophenyl, .[.pentachlorphe-.]..Iadd.pentachlorophe-.Iaddend.nyl, p-nitrophenyl and 1 -benzotriazolylof benzyloxycarbonyl-L-(N-methyl)leucine with D-alanine methyl ester toobtain the dipeptide of formulaZ--L--(N--Me)Leu--D--Ala--.[.(N--Me)Leu--.]. .Iadd.OMe;.Iaddend.treating said last-name compound with hydrogen and a noblemetal catalyst and isolating the dipeptide of formulaH--L--(N--Me)Leu--D--Ala--OMe, reacting said last-named compound with anactivated ester as defined herein of benzyloxycarbonyl- L-proline andisolating the tripeptide of formulaZ--L--Pro--L--(N--Me)Leu--D--Ala--OMe; treating said last-named compoundwith ammonia and isolating the tripeptide of formulaZ--L--Pro--L--(N--Me)Leu--D--Ala--NH₂ ; treating said last-namedcompound with hydrogen and a noble metal catalyst and isolating thecorresponding tripeptide derivative of formula 1 in which R¹ is CH₃, R²is CH₂ CH(CH₃)₂, R³ is NH₂ and Y is the amino residue D--Ala.
 16. Apharmaceutical composition comprising an acid addition salt of acompound .[.of formula 1.]. as claimed in claim 2 and a pharmaceuticallyacceptable carrier.
 17. A pharmaceutical composition comprising an aminoprotected derivative of a .[.copound of formula 1.]. .Iadd.compound.Iaddend.as claimed in claim 3 wherein the protecting group is selectedfrom the group consisting of benzyloxycarbonyl t-butoxycarbonyl, α,α-dimethyl-3,4-dimethoxybenzyloxycarbonyl, triphenylmethyl and benzyland a pharmaceutically acceptable carrier.
 18. A method of treatingParkinsonism in warm-blooded animals comprising the oral administrationthereto of 0.25-100 mg/kg of .[.an acid addition salt of.]. a compound.[.of formula 1.]. as claimed in claim 1 or a pharmaceuticallyacceptable acid addition salt thereof.
 19. A method of treating mentaldepression in warm-blooded animals comprising the oral administrationthereto of 0.25-100 mg/kg of a compound .[.of formula 1.]. as claimed inclaim 1 or a pharmaceutically acceptable acid addition salt thereof. 20.A method of treating Parkinsonism in warm-blooded animals comprising theparenteral administration thereto of 0.05-20 mg/kg .[.of an acidaddition salt.]. of a compound .[.of formula 1.]. as claimed in claim 1or a pharmaceutically acceptable acid addition salt thereof.
 21. Amethod of treating mental depression in warm-blooded animals comprisingthe parental administration thereto of 0.05-20 mg/kg .[.of an acidaddition salt.]. of a compound .[.of formula 1.]. as claimed in claim 1or a pharmaceutically acceptable acid addition salt thereof.